Important Books & Reports

Glyphosate/Roundup, falsely claimed by Monsanto to be safe and harmless, has become the world’s most widely and pervasively used herbicide; it has brought rising tides of birth defects, cancers, fatal kidney disease, sterility, and dozens of other illnesses - more

Ban GMOs Now - Dr. Mae-Wan Ho and Dr. Eva Sirinathsinghji

Health & environmental hazards
especially in the light of the new genetics - more

Living Rainbow H2O - Dr. Mae-Wan Ho

A unique synthesis of the latest findings in the quantum physics and chemistry of water that tells you why water is the “means, medium, and message of life” - more

The Rainbow and the Worm - the Physics of Organisms - Dr. Mae-Wan Ho

“Probably the Most Important Book for the Coming Scientific Revolution” - more

Mass Genome Engineering

Whole microbial genomes can be changed at will simultaneously and rapidly; with large implications for safety Dr. Mae-Wan Ho

Whole genome engineering

One major way in which synthetic biology goes beyond
conventional genetic engineering is in the manipulation of entire genomes, both
in assembling it from pieces, as genome sequence data become available, and in
modifying genes scattered over entire genomes. It must be emphasized that these
techniques work as intended only in microbes where recombination between
homologous (similar) nucleic acid sequences is the rule. In plants and animals,
however, non-homologous recombination between dissimilar sequences predominates,
and it is very difficult to target genes precisely. That is why genetic
modification of crops and livestock is inherently uncontrollable and hazardous,
as people like me have been stressing since it all began; and we have been
proved right (see [1] GM
is Dangerous and Futile, SiS 40). Synthetic biologists must proceed with
caution in targeting plant and animal genomes, because the unpredictability and
potential hazards are multiplied many times over.

The motivation for genome engineering,
according to researchers Peter Carr at Massachusetts Institute of Technology,
and George Church at Harvard Medical School, Cambridge, Massachusetts [2], is
to understand through building, to produce medicines and biofuels with a genome
optimized for the purpose, for example, to use microbes as biosensors, for
bioremediation, to hunt and destroy cancer cells; to instruct our own cells to
minimize the risk of septic shock; to produce organisms with fundamentally
altered codon usage, which could prevent an engineered lab strain using
acquired genes, and donating its engineered features to wild organisms. Or
simply, the motivation is “build to creatively explore.”

Examples
of genome engineering include Craig Venter Institute’s construction of the
first microbial genome from commercially available cassettes [3]; the deletion
of many large segments of E. coli genome to get rid of unstable DNA
elements; the transfer of much of one Archean bacteria genome into eubacteria
genome; decomposing the T7 bateriophage genome into many reconfigurable
modules; making large numbers of targeted changes to a genome simultaneously
(see below); and developing a purified translation system useful for in
vitro prototyping of genetic functions without requiring moving genes into
living cells. The manipulation of DNA segments >100 kbp in the first three
examples relied heavily on in vivo recombination techniques.

Super-exponential growth in DNA technologies

One key technique that made it possible to manipulate entire
genomes is the synthesis of DNA from scratch. Since the first synthetic gene
was produced in the 1970s, the size of synthesized DNA increased exponentially
until the early 2000s, when it experienced super-exponential growth with the
construction of the entire 582 960 bp genome of Mycoplasma genitalium
(see Figure 1a) [2]. Also since the early 2000s, the cost of sequencing and
synthesis of DNA has dramatically decreased (see Figure 1b). It is possible, by
2009, to produce DNA at 100kbp/dollar, and sequence 1 Mbp/dollar (top two
curves, Fig. 1b). But getting a novel dsDNA (double stranded DNA) gene
construct to work by design is a nontrivial process, and that is lagging far
behind (bottom curve, Fig. 1b).

“Genome engineering is in its infancy.” Carr
and Church said [2]. The new techniques have enabled initial work to be done,
but more sophisticated tools are needed at all stages: “design, DNA
construction and manipulation, implementation and testing, and debugging.”

Figure 1 Growth in DNA technologies since genetic
engineering began in the 1970s

The preferred procedure for genome manipulation is variation
and selection instead of specific design, a kind of super-accelerated
evolution. This is where mass genome engineering comes in, which has two big
potential advantages: the preexistence
of highly evolved modules in cells and microorganisms, and the possibility of
introducing combinatorial and other genome-wide changes in the lab with high
throughput techniques. One general category focuses on improving existing
functions or selecting for new functions through directed evolution.

Computer-aided design (CAD) tools for high
level design and simulation are crucial, as well as the detailed layout and
sequences of oligonucleotides for multiplex assembly of genes or genomes. CAD specifies
the complete combinations of genetic modifications for metabolic engineering
and for sequence-based screening, where the number of changes to be made is too
large, such as the genome-wide codon conversion in E. coli, where all
TAG stop codons are to be converted to TAA (another stop codon). CAD tools are
also needed to generate metabolic and signaling pathways, including processes
not yet found in nature. A major goal for the future is to automate and
integrate various aspects [2] “from protein design to compatibility of
standards and intellectual property.”

DNA synthesis companies generally use
fluid handling robots and moderately high density (96-. 384- or 1 536-well)
reaction plates, which can produce megabase pairs of DNA per month. Another
approach is microfluidic processing, which minimizes reagent and consumables
and depend on parallel synthesis of shorter sequences that are assembled into
longer ones.

MAGE for large-scale programming and accelerated
evolution of genomes

Multiplex automated genome engineering (MAGE) is being
developed for the large-scale programming and evolution of cells [3]. MAGE
simultaneously targets many sites on the chromosome for modification in a
single cell or across a population of cells, thus producing vast combinations
of genomic diversity to select for the optimum combinations. The process is cyclical
and scalable. The research team led by George Church constructed prototype
devices that automate MAGE to facilitate rapid and continuous generation of mutations.
MAGE was used to optimize the 1-deoxy-D-xylulose-5-phosphate (DXP) biosynthesis
pathway in E coli to overproduce lycopene. Mediated by the bacteriophage
l-Red ssDNA-binding protein b, gene replacement is achieved in the E.
coli strain by directing ssDNA oligonucleotides to the lagging strand of
the replication fork during DNA replication. The ssDNA were introduced by
electroporation.

Twenty genes, documented to increase lycopene yield were
targeted in an attempt to optimize translation by 90 base oligonucleotides
containing degenerate ribosome binding site sequences flanked by homologous
regions on each side. In addition, four genes from secondary pathways were
targeted for inactivation by replacement sequences with stop codons to further
improve flux through the DXP pathway. Thus, a total of 24 genes were optimized simultaneously
for lycopene production.

As
many as 15 billion genetic variants were generated. Screening was done by isolating
colonies that produced intense red pigmentation (of lycopene) on agar plates.
Sequencing of six variants revealed ribosomal binding site consensus
Shine-Dalgarno sequence (which effectively recruits ribosome to start
translation) in genes located at the beginning and end of the biosynthesis
pathway; and gene knockouts from secondary pathways. The yield of lycopene was
up to 9 mg/g dry cell weight.

In
a later application using a general co-selection strategy with genetic markers
within ~500kb of the targeted sites, the simultaneous modification of 80 sites
in the E. coli genome was achieved [4].

Is
it safe?

MAGE
combines targeted genome engineering with selection to rapidly create novel
genomes with the required characteristics, and is the most effective genome
modification process on the horizon. Such massive changes to genomes are bound
to have unintended effects through gene, epigenetic and metabolic interactions,
and it is vitally important that the new strains created are thoroughly
characterized and risk assessed.

Although
the process takes place presumably under contained use, how strict is the
containment? Are transgenic wastes – containing tens of billions of new genomes
created at any one time – discharged into the environment? These new genomes
would offer numerous opportunities for horizontal gene transfer and
recombination to create yet further new strains of bacteria and viruses, at
least some of which may be serious pathogens. It is crucial to prevent this
from happening.

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There are 2 comments on this article so far. Add your comment above.

Warren Brodey M.D. Comment left 22nd October 2012 19:07:18Have we enough wisdom to control the powerful tools we have created? Ask yourself and your family, your close friends and then your colleagues When the cost of an error is too high should I limit my creativity?
Is this a new question?
I believe it is.
Our power to destroy humanity has a new dimension. We can succeed.

Harold Titsonbeli sr Comment left 23rd October 2012 20:08:07Thank you for this,it makes connections possible to emerging diseases.We only find out what these mad scientists are doing after consideral time has past.Where is the power for an international ethics review?I am a very curious character,I always run in to unintended effects,some minor at first,however hindsight knowledge requires one to exist to review info.The benifits of this science is only to carry out depop.So it must be agreed upon by all partys' involved,not just those in the loop.My understanding of progress,wide diffinition,is that yields of modifications are based on best enviormental factors,not adversity.This comes to a bad end with gm s having a greater demand on food and water for optimum yeilds,so what happens in adverse conditions,meaning greatest time of need,drought?massive die off.This knowledge is at the second meta level,what about the next. Meta levels???? There is no impending doom to justify this effort ,other than prepare organisms to survive on ther planets ,or drastic ,maybe intended,changes to our biom